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Search: Pim-3[Title] AND Regulates[Title] AND Stemness[Title] AND Pancreatic[Title] AND Cancer[Title] AND Cells[Title] AND via[Title] AND Activating[Title] AND STAT3[Title] AND Signaling[Title] AND Pathway[Title]

Abstract

Due to its aggressiveness and unusual resistance to conventional therapies, pancreaticcancer is a highly lethal gastrointestinal malignancy with poor prognosis. According to the cancer stem cell hypothesis, there exists a fraction of cancercells, that is, cancer stem cells, responsible for tumor maintenance and therapeutic failure. Herein we investigated the involvement of proto-oncogene Pim-3 in driving the stemness properties in pancreaticcancer. Expression levels of several stemness-associated markers were examined in several pancreaticcancer cell lines. The double positive (CD24+ESA+) and double negative (CD24-ESA-) pancreaticcancercells were isolated from PANC-1 and L3.6pl, and their self-renewal ability, tumorigenicity as well as sensitivity to gemcitabine were then evaluated. Results showed that there existed heterogeneity in expression levels of stemness-associated surface markers among pancreaticcancer cell lines. CD24+ESA+ pancreaticcancercells exhibited increased tumorigenicity and decreased chemosensitivity to gemcitabine as compared to CD24-ESA- cells. Besides, the double positive (CD24+ESA+) subpopulation also exhibited greater expression level of Pim-3 when compared with the double negative (CD24-ESA-) ones. Furthermore, silencing of Pim-3 in pancreaticcancercells leads to decreased proportions of both single positive (CD24+ and ESA+) and double positive (CD24+ESA+) pancreaticcancercells. Overexpression of Pim-3 was associated with increased levels of some stemness-associated transcription factors (STAT3, etc.). Moreover, the phosphorylation level and transcriptional activity of STAT3 were decreased in Pim-3 silenced pancreaticcancercells and restoration of its activity results in restitution of stem cell-like phenotypes. Therefore, Pim-3 maintains stemness of pancreaticcancercellsviaactivatingSTAT3signalingpathway and might be used as a novel therapeutic target in pancreaticcancer.

KEYWORDS:

Expression levels of several stemness-associated surface markers in PC cell lines. A, CD133, CD24, CD34, CD44 and Pim-3 expression in human PC cell lines were detected by immunoblotting. β-actin was used as internal control. B, PC cells were incubated with fluorochrome-conjugated CD133, CD24, CD44 and ESA antibodies and subjected to flow cytometry analysis. Cells expressing specific markers were drawn in the rectangle gate as well as their percentage in total cells.

CD24+ESA+ PC cells exhibit gemcitabine chemoresistance. A and B, CD24+ESA+ PC cells were less sensitive to gemcitabine treatment. The double positive and double negative PANC-1 cells and L3.6pl cells were treated with increasing concentrations of GEM for 72h respectively. Cell viability was determined by CCK-8. Data are expressed as the mean ± SD. The experiments were repeated three times. **P < 0.01, ***P < 0.001. The percentage of CD24+ESA+ PC cells increased by GEM treatment. C and D, Expression levels of CD133, CD24, CD44 and ESA in GEM treated L3.6pl (4 μM) and MIAPaCA-2 (2 μM) analyzed by flow cytometry. Cells expressing specific markers were drawn in the rectangle gate as well as their percentage in total cells.

Pim-3 induces expression of stemness-associated markers in PC cells. A, Expression level of CD24, CD44 and ESA in PANC-1-Pim-3-sh and MIAPaCA-2-Pim3 analyzed by flow cytometry. Single positive cells (CD24+, CD44+ and ESA+) and their percentage in total cells were drawn in the rectangle gate. CD24+ESA+ cells were shown in the first quadrant as well as their percentage in total cells. B, Expression of other stemness-associated markers were examined in PANC-1-Pim-3-sh, MIAPaCA-2-Pim-3 and their parent cells at protein level by immunoblotting and C, mRNA level by quantitative RT-PCR (mean ± SD; n=3; *P<0.05, **P<0.01, ***P<0.001). D, Expression of Pim-3 and other stemness-associated markers were examined in CD24+ESA+ and CD24-ESA- PANC-1 cells at protein level by immunoblotting and mRNA level by quantitative RT-PCR (mean ± SD; n=3; *P<0.05, **P<0.01, ***P<0.001). E, Colonies formed by PANC-1-Pim-3-sh, MIAPaCA-2-Pim-3 and their parent cells. Data are presented as the mean ± SD. The experiments were repeated three times. **P<0.01.

STAT3 mediates Pim-3-induced stemness in PC cells.A, Expression level of pSTAT3 and total STAT3 were detected by immunoblotting in PANC-1-Pim-3-sh, MIAPaCa-2-Pim-3 and their parent cells as well as M-110 treated PANC-1 and MIAPaCa-2-Pim-3. β-actin was used as internal control. B, Transcriptional activity of STAT3 in PANC-1-Pim-3-sh, MIAPaCa-2-Pim-3 and their parent cells. Cells were co-transfected with a mixture of the STAT3 luciferase reporter and Renilla plasmid. After 48 h, cells were harvested in passive lysis buffer and luciferase activity was assessed using a dual-luciferase assay system. Data are presented as normalized relative luciferase activity (mean ± SD; n = 3, **p < 0.01). C, Expression levels of stemness-associated markers in MIAPaCa-2-Pim-3-STAT3-si and PANC-1-Pim-3-sh-STAT3cells. MIAPaCa-2-Pim-3 was transiently transfected with STAT3 siRNA and PANC-1-Pim-3-sh was transiently transfected with STAT3 plasmid. Expression level of pSTAT3 and total STAT3 were detected by immunoblotting in MIAPaCa-2-Pim-3-STAT3-si and PANC-1-Pim-3-STAT3cells. β-actin was used as internal control. D, Expression level of CD24, CD44 and ESA in MIAPaCa-2-Pim-3-STAT3-si and PANC-1-Pim-3-sh-STAT3 were analyzed by flow cytometry. Single positive cells (CD24+, CD44+ and ESA+) and their percentage in total cells were drawn in the rectangle gate. CD24+ESA+ PANC-1-Pim-3-sh cells were shown in the first quadrant as well as their percentage in total cells.